The long-term objectives of this research are to use the special opportunities available in Saccharomyces and related species to bring an understanding of epigenetic processes to a much deeper level, exploiting innovative approaches developed in the present review period in ways that are inaccessible in other organisms. The PI has developed an assay that captures the transient and temporary expression of epigenetically silenced genes, making a permanent and heritable mark of that transient expression. This assay will be used to identify comprehensively every genetic contribution to the stability of epigenetic silencing, which has never before been possible. The assay will be adapted to allow a critical test of the leading model for the inheritance of silenced chromatin. New data at much higher resolution than that of earlier studies have called into question all previous ideas of how heterochromatin, or other chromatin structures, spread from sites of nucleation. Systematic evaluation of the structure of silenced chromatin as it is formed will test whether existing models can be adapted, or need to be replaced. In keeping with the emerging importance of metabolic intermediates as mediators of epigenetics and cancer, the PI will resolve the paradoxical role of Sir2, the founding member of the sirtuin family of deacetylases, with respect to the potential role of 2-O-acetyl-ADP-ribose in silencing. The mechanism(s) operating to repress gene expression in silenced chromatin are not yet known. The PI will use the RNA polymerase from phage T7 to dig deep into the mechanism(s) of repression in silenced chromatin, along with tests of the contribution of specific transcription factors to the silencing process. Nutrition is the single most important translational interface between the environment and the epigenome, with folate playing a central role. The impact of folate limitation on the structure of yeast and human epigenomes will be determined to reveal the extent and mechanism by which nutritional limitation alters the expression of the genome. Finally, a newly developed yeast species, optimally chosen for the purpose, will be used to learn how Sir-based silencing and RNAi-based silenced cooperate or compete, and then separated during speciation.